The purpose of the present study was ; plasma [Na+] increased by 1.2% (p < 0.0001). Haematocrit decreased (p = 0.0005). The volume of the feet remained unchanged (p > 0.05). Plasma volume and urine specific gravity increased (p < 0.0001). Fluid intake was positively related to the change in the volume of the feet  and negatively to post-race plasma [Na+] . Running speed was negatively related to both fluid intake  and the change in feet volume . The change in the volume of the feet was negatively related to the change in plasma [Na+] . The change in body mass was negatively related to both post-race plasma [Na+]  and running speed .Body mass decreased by 1.8 kg (2.4%) (An increase in feet volume after a 100-km ultra-marathon was due to an increased fluid intake. Participation in ultra-marathon running is of increasing popularity -5 where +]). A decreased serum [Na+] as well as an increase in total body water has also been reported for male 100-km ultra-marathoners [+] remained unchanged in female 100-km ultra-marathoners while drinking ad libitum , fluid retention and an increased shift of fluid from the intracellular to the extracellular fluid compartment. Similar findings on fluid homeostasis have been reported for five subjects during seven consecutive days of hill-walking, where facial and ankle oedemas also due to a retention in plasma [Na+] and a shift of water from the intracellular to the extracellular space with an expansion of the extracellular volume have been observed [+] retention  [+] has been reported in several studies [+], indicating that exercise-associated hyponatremia (EAH) is associated with minimal body mass loss or body mass gain [+] < 135 mmol/l during exercise or up to 24 h after exercise [+] retention due to an increased aldosterone activity, additional mechanisms could lead to a retention in total body water in ultra-endurance athletes such as protein catabolism and subsequent development of hyperproteinemic oedemas  -21, an i+] . An inve studies ,20,22-26ass gain . This isexercise . Since u oedemas , an incr oedemas ,30, an i oedemas  or impai oedemas ,12.®. The authors presumed that these disparate findings were due to a redistribution of the limb volume limited to hands and feet and not involving the whole limb  and feet volume and that an increased fluid intake would lead to both an increase in feet volume and a decrease in plasma [Na+], thus leading to an increased prevalence of EAH. To test this hypothesis, we investigated a potential association between changes in feet volume using plethysmography with fluid intake in male 100-km ultra-marathoners.Basing upon these recent findings, we might assume that an increased fluid intake may lead to an increase in feet volume. To our knowledge, there have been no studies to date investigating a potential association between changes in the feet volume and fluid intake in a 100-km ultra-marathon. The aims of the present study were, therefore, to investigate in 100-km ultra-marathoners . Standardisation of posture prior to blood collection was respected since postural changes can influence blood volume and therefore haematocrit  increased significantly (p < 0.0001) by 1.2% from 137.0 (2.7) mmol/l to 138.6 (2.67) mmol/l, with a mean difference of 1.6 (3.1) mmol/l. Pre-race, 10 subjects showed plasma [Na+] < 135 mmol/L with values between 131 mmol/L and 134 mmol/L. Post-race, four subjects (5.3%) developed asymptomatic EAH with post-race plasma [Na+] between 132 mmol/L and 134 mmol/L. The lowest post-race plasma [Na+] was 132 mmol/L in these subjects. Pre-race plasma [Na+] in these four subjects was 139 mmol/L. Table +] < 135 mmol/L, with a pre-race plasma [Na+] of 133 mmol/l in one subject, and 131 mmol/L in the other subject, respectively. The change in body mass was significantly and negatively related to the change in plasma [Na+] , but showed no association with the change in plasma [Na+].The subjects consumed a total of 7.64 2.85) L of fluids during the run, equal to 0.63 (0.20) L/h or 0.10 (0.03) L/kg body mass, respectively. Fluid intake varied between 2.7 L and 20 L  developed asymptomatic EAH with plasma [Na+] between 132 and 134 mmol/L.The first aim of the present study was to investigate whether peripheral oedemas leading to an increase of the feet volumes would occur in 100-km ultra-marathoners. Based on the existing literature, we hypothesized that an ultra-marathon can lead to peripheral oedemas with an increase in the feet volume and that fluid overload would be associated with these increases. In case of fluid overload leading to an increase in the feet volume we hypothesized furthermore finding an association between changes in plasma  [+] increased. In case of dehydration, as has been demonstrated in 12- and 24-hour ultra-marathoners [+] is expected when the water loss, including water loss by sweat, inducing dehydration is hypotonic compared with plasma  -21, an i+] . In the athoners , both a athoners ,37. Furth plasma . Howeverh plasma . Rather h plasma , both Sph plasma  and Rogeh plasma  suggesteh plasma  concludeh plasma ,40 suggeh plasma  and not +] were maintained. Thus, Hew-Butler et al. [+] and not body mass during exercise. In addition, Nolte et al. [+] increased from 137.0 (2.7) mmol/l to 138.6 (2.67) mmol/l. Although the 1.6 (3.1) mmol/l increase in plasma [Na+] from pre-race to post-race was statistically significant, plasma [Na+] was still maintained within the normal range limits (135-145 mmol/L) [+] increased in the present subjects, this could be a possible reason for the increase in plasma volume. However, the change in plasma volume showed no correlation with the change in plasma [Na+] in the present subjects, but was associated with fluid intake. Presumably, the increase in plasma volume was due to fluid ingestion and there may be a potential internal water source, for example water previously stored with glycogen, that can be released during exercise and maintain blood biochemical parameters despite an absolute body weight loss [+] and circulating blood volume and not body mass during prolonged endurance exercises and that a change in body mass during exercise may not reflect exact changes in the hydration status  between 132 and 134 mmol/L. Pre-race plasma [Na+] in these four subjects was 139 mmol/L. Two athletes showed plasma [Na+] < 135 mmol/l both pre-and post-race. By definition, no EAH occurred in this two subjects, since they both had a pre-race plasma [Na+] < 135 mmol/L. Overall, 10 subjects showed plasma [Na+] < 135 mmol/L with values between 131 mmol/L and 134 mmol/L pre-race. No symptomatic EAH occurred. The prevalence of 5.3% subjects with asymptomatic EAH in these 76 ultra-marathoners is rather low compared to other studies reporting prevalence of EAH in marathons and ultra-marathons between 0% and 51.2% [+] and the change in body mass; athletes who lost the least weight or even gained weight, had the lowest plasma [Na+] post- race. Our finding corresponds to results in several former studies [+].A further finding was that four runners (5.3%) developed asymptomatic EAH with post-race plasma . This result supports the findings of the existing data that EAH is associated with fluid overload [+] of 136 mmol/L and the foot volume decreased by 53%. He was also among the fastest runners finishing within 582 min (9 h 42 min). This result is not in line with our and other findings that a high fluid intake is correlated with lower post-race plasma [Na+]  -21. Possma [Na+]  or inabi+]. As fluid intake was associated with the change in the foot volume, an increased fluid intake generally led to both a decrease in plasma [Na+] and an increase in the foot volume. Obviously, slower runners were drinking more and their post-race plasma [Na+] tended to decrease, since both fluid intake and the change in the feet volume was significantly and negatively related to running speed. In addition, slower runners showed an increase in the foot volume. Presumably, slower runners were sweating less and drinking at a higher rate than were the faster runners. As slower runners are more likely to overconsume fluids . Therefore, we concluded that fluid overload occurred in slower runners and was responsible for the development of oedemas in the foot. In addition, post-race plasma [Na+] decreased in those runners. Our data support the finding that fluid overload is the main risk factor for developing EAH [In summary, this study demonstrated that fluid intake was positively related to the volume of the foot in 100-km ultra-marathoners. An increase in the foot volume occurred in athletes with an increased fluid intake. In addition, slower running speed was associated with an increase in the foot volume and the change in foot volume was negatively correlated to the change in plasma [Naping EAH -21. For ping EAH -21 and cThe authors declare that they have no competing interests.CC wrote the manuscript, BK and PK collected the data at the race, CAR and TR assisted in data analysis, data interpretation and manuscript preparation. All authors have read and approved the final version.


Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen  Staphylococcus aureus is normally a harmless commensal bacterium residing in 25% of the adult population, yet is a dangerous human pathogen in susceptible individuals [S. aureus is also notorious for its capacity to develop resistance against antibiotics. For example, methicillin- and vancomycin-resistant strains of S. aureus are among the major causes of nocosomial and community infections in the USA [S. aureus and other dangerous pathogens [ividuals ,2. S. au the USA ,4. We arS. aureus [S. aureus to persist in the bloodstream of the infected host. More than 70% of clinical isolates of S. aureus belong to either the CP5 or the CP8 serotypes [The biosynthetic machinery that generates the CP (capsular polysaccharide) is absent in humans, and represents an attractive target to fight . aureus –8. CP foerotypes .N-acetyl-L-fucosamine, N-acetyl-D-fucosamine, and N-acetyl-D-mannosamine uronic acid. The UDP-L-FucNAc (UDP-activated form of N-acetyl-L-fucosamine) is synthesized from its glucosamine precursor (UDP-D-GlcNAc). This reaction has been described in S. aureus and several other pathogenic bacteria possessing CP such as Streptococcus pneumoniae and Bacteroides fragilis [The basic structure of CP of serotypes CP5 and CP8 consists of alternating units of three types of monosaccharides: fragilis ,11. L-Fufragilis ,13.S. aureus [Helicobacter pylori displays 40% homology to CapE and catalyses the same enzymatic reactions [The transformation of UDP-D-GlcNAc in UDP-L-FucNAc requires three enzymes  in . aureus ,14. Thes. aureus : a N-ter. aureus , and a C. aureus . A previ. aureus . The twoeactions ,19. HoweHerein we present the first crystal structure of CapE, revealing that this enzyme forms a stable and functional homo-hexamer. From the comparison of three crystal structures, we identified a novel and dynamic element unique to the capsular-polysaccharide synthesizing proteins that we called the latch. The special configuration of the active site of CapE, together with the unique features of the latch, exposed a distinctive group of enzymes of bacterial origin within the SDR superfamily sharing these two special elements.3-GlcNAc (6′-Azido-6′-deoxy-UDP-N-acetylglucosamine) was carried out as previously described [The synthesis of UDP-6Nescribed .6 tag at the N-terminus was expressed in Escherichia coli BL21 (DE3) cells, and purified as described previously [ϵ=19200 M−1 cm−1). Muteins of CapE were prepared with a Quick-Change kit following the instructions of the manufacturer. Protein expression and purification were carried out as above.CapE with a Hiseviously ,21. Afte3 within 1 week. Crystals were transferred to a cryoprotectant solution consisting of mother liquor supplemented with 25% (v/v) glycerol, plunged into liquid N2 and stored until data collection.Crystals of CapE suitable for X-ray diffraction analysis were obtained by the hanging drop method by mixing 1 μl of fresh protein solution at 8 mg/ml and 1 μl of crystallization solution. Crystals of wild-type CapE with coenzyme bound were obtained in 0.72 M sodium succinate (pH 7.0) and 1.4 M potassium formate. Rod-shaped crystals grew to an approximate size of 30×30×100 μm3-GlcNAc. Extensive screening with an Oryx8 robot (Douglas Instruments) [2SO4. For freezing, single crystals were passed through a small drop of paratone (Hampton Research) and plunged in liquid N2. Crystals of mutein K126E were obtained in 100 mM Hepes (pH 7.5), 2% (v/v) PEG400 and 2.0 M ammonium sulfate. Suitable crystals were soaked in mother liquor supplemented with 25% (v/v) glycerol and 500 μM substrate (the substrate was not observed in the electron density) and stored in liquid N2.Crystals of CapE with substrate analogue were obtained by the co-crystallization method using 100 μM UDP-6Nruments)  yielded H. pylori (PDB entry code 2gn4) as the search model. Coordinate refinement was carried out with PHENIX [Suitable crystals of CapE were mounted under a stream of cold nitrogen (100 K) at beamlines BL5A and AR-NE3A of the Photon Factory . Data were processed with the program MOSFLM  and mergh PHENIX , REFMAC5h PHENIX  and COOTh PHENIX . Data weh PHENIX . The strh PHENIX . Data coEnzymatic activity of CapE was monitored by the method described in Miyafusa et al. . In a ty−1. The buffer baseline was subtracted from the protein thermogram and the data subsequently were normalized by protein concentration and adjusted to a two-stage unfolding curve [The thermal stability of CapE was determined in a VP-capillary microcalorimeter . Proteins at a concentration of 18 μM were equilibrated in a solution containing 50 mM Hepes (pH 7.4) and 150 mM NaCl. Thermograms were recorded between 283 K and 373 K at a rate of 1 K minng curve  using th3-GlcNAc.We determined three crystals structures of CapE by X-ray diffraction methods at resolutions 2.1–2.8 Å . Two strHelicobacter pylori (40% identity), which is also the closest structural homolog of CapE in the protein data bank . We note that the enzyme following CapE in the biosynthetic route of UDP-D-FucNAc in S. aureus, CapF, has the lowest similarity score among the first 950 structural hits found with the program DALI [A three-dimensional homology search in the DALI server  indicateram DALI .latch because it engages two molecules of CapE in the crystallographic dimer. The latch is not observed in the two crystal structures of CapE with coenzyme bound because of dynamic disorder. In contrast, the latch is clearly observed in the structure with substrate analogue bound (see below). We note that the tertiary structure of CapE and FlaA1 differs significantly from each other at the latch region . Whereas the latch connects two protomers of CapE of the hexameric complex, the equivalent loop of FlaA1 (residues 292–318) folds into its own protein chain.CapE consists of three well-defined regions: a Rossmann domain for binding the coenzyme NADPH, a substrate binding domain, and a 23-residue loop peripheral to the protein core A and 2B.3-GlcNAc is clearly visible in the electron density map . In particular, the UDP moiety of the substrate analogue establishes numerous H-bond and electrostatic interactions with polar residues of the protein.The substrate-binding domain consists of residues 168–199, 227–260 and 310–337. The substrate-analogue UDP-6Nsity map A. The elsity map B. The come FlaA1  (Supplemhttp://www.bioscirep.org/bsr/033/bsr033e043add.htm). A detailed view of the local environment around the coenzyme is depicted in The coenzyme-binding region is the largest functional element of CapE. This domain adopts a classical Rossmann fold ,35, comphttp://www.bioscirep.org/bsr/033/bsr033e043add.htm) [Analysis of the crystallographic symmetry reveals that CapE is a homo-hexamer in the crystal form . The proadd.htm) .2 of surface area upon oligomerization, i.e. 24% of the total solvent-exposed surface. Each protomer of CapE interacts with adjacent molecules through three major interfaces   . This inhttp://www.bioscirep.org/bsr/033/bsr033e043add.htm). hUDGH (human UDP-α-D-glucose dehydrogenase) is a well-studied hexameric enzyme showing large structural changes during its catalytic cycle [The A–B and A–C surface areas of CapE in complex with coenzyme are significantly smaller than that of CapE with substrate analogue bound. The A–B and A–C surface areas calculated from the coordinates of CapE with coenzyme bound are reduced by 60 and 65%, respectively . The reaic cycle ,42. The The Rossmann domain of CapE is located at the centre of the hexamer and mediates most of the inter-chain contacts, whereas the substrate-binding domain is found at the periphery of the complex . This ar256 and Gly252 (6.0 Å). Although these observations must be understood within the context of the crystal structure, they are suggestive of a significant mobility at the substrate domain during catalysis.The crystals structures of CapE with analogue bound or with coenzyme bound were compared with the program DYNDOM  to quanthttp://www.bioscirep.org/bsr/033/bsr033e043add.htm). The latch connects two contiguous subunits of CapE, and forms a large fraction of the A–B and A–C interface of the hexamer with very high shape complementarity (SC=0.79 [http://www.bioscirep.org/bsr/033/bsr033e043add.htm). Together with the residues of neighbouring subunits of CapE, the latch contributes >3500 Å2 of buried surface area, including 28 H-bonds. The interaction between the latch and the adjacent protomer is also held together by contacts involving four large hydrophobic residues: Leu288, Tyr290, Tyr293 and Ile301. In addition, the latch occupies a strategic position at the entrance of the substrate-binding pocket, potentially regulating the access of the substrate to the active site  . Serotypes of a majority of these bacteria are encapsulated, suggesting a direct association between this novel group of SDR enzymes, the production of CP and pathogenesis [A search of tertiary structure homologues of the latch with the DALI server  did not ogenesis ,12,45–49134xxxK138 without the catalytic tyrosine. Importantly, the sequence alignment of the family of proteins sharing the latch (see http://www.bioscirep.org/bsr/033/bsr033e043add.htm). In contrast, the other SDR structural homologues do invariably possess the canonical YxxxK motif . We propose that the unique structural element that we called the latch, and the unique composition of the active site residues define a novel subfamily of polysaccharide-synthesizing enzymes within the SDR super-family of proteins.The coenzyme-binding domain hosts the canonical catalytic triad, which invariably contains the sequence YxxxK in SDR enzymes . Strikingly, the corresponding sequence of CapE is altered to Match see D demonsthttp://www.bioscirep.org/bsr/033/bsr033e043add.htm). No excess coenzyme was added in the enzymatic assay because CapE was purified in complex with the coenzyme. The level of NADPH bound to CapE was essentially constant among all muteins as estimated from the ratio Abs260/Abs280 (Supplementary Table S3). Firstly, individual residues of the active site and substrate-binding pocket were mutated: Asp125, Lys126, Met134 and Glu257 were changed to Ala . All these individual muteins exhibited a much-diminished activity compared with the wild-type enzyme. The muteins K126A, K126E and E257A where completely inactive, demonstrating the importance of these residues for the functional binding of the substrate. Meanwhile the activity of D125A and M134A was strongly impaired (4–6-fold lower conversion than WT protein). We note that all the muteins eluted as hexamers during their final step of purification by SEC.To strengthen the structural analysis we prepared several muteins of CapE by site-directed mutagenesis  by mutating separately five different residues to Ala: Phee enzyme A. Howevee enzyme B. SimilaS. aureus belonging to the biosynthetic pathway of CP. CapE forms a robust hexamer held together by three different interfaces, A–B, A–F and A–C. The largest A–B interface contains a mobile motif that we denominated the latch. The latch is a flexible element that increases the contact surface area of adjacent monomers of CapE in the presence of the substrate analogue UDP-6N3-GlcNAc. The structural analysis also demonstrates a rotation of the substrate-binding domain when the substrate analogue binds to the enzyme. Importantly a sequence alignment of the latch, and of the active site residues of CapE, has identified a novel subfamily of SDR enzymes involved in the synthesis of polysaccharide of Gram-positive and Gram-negative pathogenic bacteria.We determined the first set of crystal structures of the enzyme CapE of


Stroke contributes significantly to disability and mortality in developing countries yet little is known about the determinants of stroke outcomes in such countries. 12% of Malawian adults have HIV/AIDS. It is not known whether having HIV-infection alters the outcome of stroke. The aim of this study was to document the functional outcome and mortality at 1 year of first-ever acute stroke in Malawi. Also to find out if the baseline variables, including HIV-infection, affect the outcome of stroke.147 adult patients with first-ever acute stroke were prospectively followed up for 12 months. Conventional risk factors and HIV-infection were assessed at baseline. Stroke severity was evaluated with modified National Institute of Health Stroke Scale (mNIHSS) and functional outcome with modified Rankin scale (mRS). Fifty (34%) of patients were HIV-seropositive. 53.4% of patients had a poor outcome  at 1 year. Poor outcome was related to stroke severity and female gender but not to presence of HIV-infection. HIV-seropositive patients were younger and had less often common risk factors for stroke. They suffer more often ischemic stroke than HIV-seronegative patients.Mild stroke and male gender were associated with favourable outcome. HIV-infection is common in stroke patients in Malawi but does not worsen the outcome of stroke. However, it may be a risk factor for ischemic stroke for young people, who do not have the common stroke risk factors. Our results are significant, because stroke outcome in HIV-seropositive patients has not been studied before in a setting such as ours, with very limited resources and a high prevalence of HIV. In developing countries the number of disability-adjusted life years caused by cerebrovascular disease is more than six times higher than in high-income countries: More than 85% of all stroke deaths occurs in low-income countries There is a paucity of data on the outcome of stroke in sub-Saharan Africa. Only two reports are available, both from West Africa. One-year case-fatality of stroke in the Gambia and Senegal was 62% and 50% respectively. In the Gambia hospital mortality following a stroke was 41% Malawi is situated in Central-Southern Africa . AccordiThe primary aim of this study was to determine the functional outcome of first-ever acute stroke at one year of follow-up. Secondary aims were to describe the one-year mortality of stroke and to determine the effect of baseline demographics, including presence of HIV-infection.This was a prospective study of patients presenting with a first-ever acute stroke to the Queen Elizabeth Central Hospital (QECH) in Blantyre. QECH is the largest hospital in Malawi. It provides all levels of care, from primary to tertiary to patients within Southern Malawi. The QECH is the only public hospital within Blantyre District. Blantyre has a population of 1 million. It is the second largest city in Malawi after the capital city, Lilongwe  [12].The study recruitment took place between February 2008 and April 2009.The objectives were to determine the functional outcome of first-ever acute stroke and to determine the effect of baseline demographics in Malawi, a sub-Saharan African country with high HIV-prevalence.Patients were eligible for the study if, they were 18 years or older, had a first-ever acute stroke (0–7 days before admission), and lived within 100 km of QECH. The diagnosis of stroke was based on clinical findings, according to WHO recommendations Using information from the patient, their guardian and the patient's hand-held health record, demographic details, past medical history and medication use were recorded. If age was unknown it was estimated, within 5 years, by asking the patient's life history and age of their children. Current cigarette smoking and heavy alcohol use (estimated >100 g/day) were recorded. Study clinicians DC or TH carried out clinical examination. This included a detailed cardiovascular and neurological examination. The Mid Upper Arm Circumference (MUAC) was used to estimate nutritional status. Serum total, random cholesterol, full blood count, random or fasting capillary blood glucose , rapid plasma reagin-test to screen for syphilis (RPR), and presence of HIV-infection using two standard rapid immunoassays (Uni-Gold™ Recombigen® HIV and Determine® HIV-1/2) were measured. If the HIV-test was reactive the WHO HIV clinical stage was recorded WHO definitions were used for hypertension, diabetes mellitus and hypercholesterolemia Stroke severity at recruitment was assessed by the National Institute of Health Stroke Scale (NIHSS) Patients were followed up 6–8 weeks, six months and one year from stroke onset. At follow-up new health events, blood pressure and mRS were recorded. If possible, follow-up was done at a designated clinic at QECH. For patients who were unable to attend clinic a home visit was carried out. Alternatively the patients or their guardians were contacted by phone and the mRS was done based on information given.The Malawi College of Medicine Research Ethical Committee (COMREC P 07/07/573) granted approval for the study. The patients, or, in case of reduced consciousness or severe dysphasia, the guardians, gave consent.2 test and means by Student's t test, for normally distributed data, and Kruskal-Wallis test for non-parametric data.The primary outcome measure was mRS-score at one year, comparing a good/fair outcome (mRS 0–3) to a poor outcome (mRS 4–6). Data were analysed using Statview™ and STATA™ for multivariate analysis. Descriptive statistics  were used to describe the baseline data. Proportions were compared by XOne hundred and fifty patients were recruited. Brain imaging was available in 127: 124 patients had CT and 3 had MRI. Three patients were excluded from analysis as their brain imaging or follow-up suggested probable malignancy, so the final sample was 147 .2 = 43.1, p<0.0001). In those who were HIV-seropositive the WHO clinical staging was 1 or 2 in 39 (78.0%). Despite this, 28/35 in whom results were available, had CD4 cell counts of <300 cells/µl. Eleven subjects were taking HAART. Six had started HAART within the past 6 months, the remainder had been taking HAART for at least one year, however, three out of five patients with long term HAART use had CD4 <100 cells/µl, suggesting a possibility of HAART-failure. 100 patients had an ECG, of whom only 7 had atrial fibrillation (AF). The mean age of AF cases was 65.9±12 years and 4/7 were HIV-seropositive. Fifteen subjects had a reactive RPR suggesting syphilis infection, five of these were HIV-seropositive.The mean (±SD) age was 54.2 (±16.9) years, 47.7% were female. Hypertension, diabetes and hypercholesterolemia were present in 55.0%, 21.0% and 15.0%, respectively. Twenty-six (17.7%) admitted smoking tobacco and 22 (15.0%) were heavy alcohol drinkers. Fifty (34.0%) of patients were HIV-seropositive. They were significantly younger , had lower haemoglobin levels , and were less likely to have stroke risk factors of hypertension , and diabetes . There was a strong relationship between age at the time of first ever stroke, and HIV-status; 65.2% of those aged <55 years were HIV-seropositive whereas only 10.3% of those >55 years were HIV-seropositive  and was less common in HIV-seropositive patients . Multiple logistic regression controlling for age, gender and other vascular risk factors showed that hypertension and HIV-status were independently associated with the type of stroke; For ICH and hypertension  for HIV-seropositive and ischemic stroke . The clinical subtypes, pre-stroke functional status (mRS) and stroke severity (mNIHSS) are presented in Thirty-two  scans showed intracerebral hemorrhage (ICH). The remainder were consistent with cerebral infarction . ICH was2 = 4.4, p = 0.035) and stroke severity on admission , but not HIV. High mNIHSS score at presentation (mNIHSS ≥13) did not relate to type of stroke, hemorrhagic or ischemic . In multiple logistic regression mNIHSS≥13 at presentation  and female gender  were independent determinants of mortality at one year.Thirty-three patients (22.4%) died in hospital. Outcome at 1-year was unavailable on 16 subjects. Cumulative mortality rates at 6 weeks, 6 months, and 1-year were 37/134 (27.6%), 52/134 (38.8%), and 59/131 (45.0%), see . Mortali2 = 3.9, p = 0.048), and more severe stroke, mNIHSS ≥13, at presentation , but not with HIV-status. In multiple logistic regression mNIHSS ≥13 at presentation , and female gender  were independent determinants of a poor outcome (mRS 4–6) at 1 year.The functional outcome (mRS) at 6 weeks, 6 months and 1 year are shown in The average length of hospital stay was 15.7±10.7 days. Twenty-nine (19.7%) patients were transferred to a residential rehabilitation unit. Nine (6.1%) patients received outpatient physiotherapy. 127 (86.4%) patients were put on ASA (75 mg once a day). In 20 of these subsequent CT revealed hemorrhage and the ASA was stopped. Anticoagulants, ACE-inhibitors and statins are not routinely available so were not considered in the evaluation. Thirteen patients had another non-fatal stroke during one-year follow-up. Two patients developed secondary epilepsy and one patient developed thalamic pain syndrome. One patient was diagnosed hypertensive during follow-up. Of the HIV-seropositive patients that were not on HAART previously, 11 started it during the follow-up year.This study describes mortality and functional outcomes in a group of patients presenting with a first-ever acute stroke in a sub-Saharan African country with high HIV-prevalence. The prevalence of HIV-infection was 34%. We used a strict case definition to identify stroke patients and excluded non-stroke diagnoses on the basis of the clinical presentation and brain imaging.2Hypertension was the most prevalent vascular risk factor, and was present in 55% of patients. Diabetes was present in 21% of patients. Both of these risk factors were very much clustered in older patients. A recent survey in Malawian adults, aged 25–64 years, showed the prevalence of raised blood pressure, raised blood glucose and hypercholesterolemia were 31.6%, 5.6%, and 10.0% respectively Fifty (34%) of patients were HIV-seropositive. The average age of these patients was 39.8 years. They had less often hypertension and diabetes and more often ischemic stroke. This supports the proposed suggestion that HIV is a risk factor for ischemic stroke One-year outcome data was available on almost 90% of patients recruited. The main determinants of death, or death and severe disability at 1-year were severity of stroke on admission, and gender. A gender difference in favour of men in stroke outcome is known. In earlier reports this is reported to be due to women being older, having higher rates of AF, and being less likely to receive acute treatment This study has a few shortcomings. Recruitment was hospital-based so does not include stroke patients with mild symptoms who do not present to hospital, or the very severest strokes, who die before coming to hospital, or are kept at home. At times we were frustrated by the limitations of our setting. Equipment, particularly the CT and ECG-machine broke and could not be repaired within the time frame of the study. Hence, there is some missing data in these areas.Patients presenting with stroke in central hospital in Malawi divided into two populations. Older patients, over 55 years, were most likely to be HIV-seronegative and have “traditional” vascular risk factors. Younger patients were very likely to be HIV-seropositive and vascular risk factors were uncommon. This suggests that HIV-infection could be a risk factor for stroke for young people without common stroke risk factors. All the patients received similar post-stroke care, and although there were differences in functional ability, as reflected by the mRS-score at presentation, the 1-year outcome was not associated to HIV-status. The 1-year mortality was 40.1%. 47.6% had a poor outcome (mRS 4–6). Stroke severity on admission and female gender were the only two independent factors associated with outcome. It is also a concern that only half of those with HIV who were eligible to start HAART did so, during one year of follow up.


Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples.C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively.With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds.The  Campylobacter spp. are recognized as the leading human foodborne pathogens in developed countries [Campylobacter, the thermophilic species Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the most frequently associated with illness, accounting for over 95% of infections (respectively responsible for 80 to 85% and 10 to 15%) [Campylobacter pose an important risk for human infection from contamination of carcasses at slaughter, of milk, and water contaminated by livestock wastes and slurries [C. jejuni in animal intestinal tracts, for example, broiler chickens, cattle, and wild-living mammals and birds [Campylobacter , to exhibit high counts of this pathogen in their faeces (ranging from 102 to 107 Colony Forming Units (CFU) of Campylobacter per gram), and to show a dominance of C. coli [C. jejuni in pigs and of C. coli in chickens [Campylobacter in livestock animals.ountries ,2. Withi to 15%) . Livestoslurries -6. Reseand birds ,8. Pigs  C. coli . Neverthchickens -15. GiveCampylobacter using conventional methods is slow (culture-based methods can take up to five days) and problematic due to their fastidious growth requirements and biochemical inertness [C. coli and/or C. jejuni in complex substrates like faeces or environmental samples is difficult as the culture conditions have to be selective enough to avoid overgrowth from competiting organisms. Additionally these bacteria may enter into a viable but nonculturable state (VBNC) [Campylobacter spp., especially C. coli and C. jejuni, by phenotypic tests is difficult and hippurate hydrolysis test used to distinguish these two species is often problematic [C. jejuni may also coexist with C. coli in pigs, but at 10-100-fold lower numbers than C. coli [C. jejuni will be less frequently isolated from such samples because only a few colonies are identified to the species level with conventional culturing and biochemical testing techniques. Molecular methods are an alternative to the bacteriological method for the detection of C. coli and C. jejuni in various substrates [C. jejuni and/or C. coli in pure culture [C. coli and C. jejuni directly from pig faecal, feed, and environmental samples.The identification of nertness ,17. Moree (VBNC) . The corblematic . Further C. coli ,20, so Cbstrates -24. Real culture , in poul culture ,27, and  culture -30 and f culture -33. HoweC. coli and C. jejuni directly in pig faecal, feed, and environmental samples. The first step in the development of the assay was the definition of the multiplex PCR assay to quantify C. coli and C. jejuni isolates from bacterial cultures. These real-time PCR assays were coupled with a modified DNA extraction protocol and then examined for their ability: (i) to evaluate DNA purification with different parameters , (ii) to measure the sensitivity and the specificity of PCR-based detection in faecal samples, and (iii) to detect C. jejuni among predominant C. coli. Finally, the last step was the application of the real-time PCR assays to detect and quantify C. coli and C. jejuni in complex substrates like feed, environmental samples, and faeces from experimentally as well as naturally infected pigs. The bacterial culture was used as a gold standard for their validation.The present study aimed to develop a species-specific real-time PCR method to detect and quantify C. coli and C. jejuni was tested against different strains of C. coli (n = 77) and C. jejuni (n = 54), all of which were correctly identified. Moreover, no signal was observed for any of the other Campylobacter species tested as well as for a range of bacteria, which could be present in faecal samples or responsible for diarrhoea in pigs and humans  [Campylobacter-negative faecal, feed, and environmental samples and examined in duplicate with each real-time PCR assays produced threshold cycle (Ct) values ≥ 42 when 5 μL of extracted DNA was used as the starting template. All samples in which both duplicates had a Ct value below this threshold were regarded as positive.The specificity of each primers-probe set for the detection of ns Table . Finally. (2004) . The DNAC. coli , providing an accurate measurement over a large variety of starting target amounts . For the environmental samples, they were around 103 CFU/m2 for both species and both sampling sites . For both species, the standard curves showed linearity from about 102 to 108 CFU and 103 to 107 CFU for feed and environmental samples respectively.The detection limit for the quantitative real-time PCR assays in spiked faecal samples were 2.5 × 10s Figure , similarL Figure . For theCampylobacter-negative spiked faecal samples was subjected to each real-time PCR in ten duplicates, with 10 different mixes performed on different runs. The results are reported in Table C. coli real-time PCR and from 0.91 to 2.53% for C. jejuni real-time PCR. The standard curves were y = -3.33x + 45.82 with R2 = 0.98 for C. coli and y = -3.24x + 46.00 with R2 = 0.98 for C. jejuni. The CV of the Ct values for the ten different inter-assay experiments, including the DNA extraction procedure, ranged from 0.57 to 2.58% and from 0.70 to 2.10% respectively for C. coli and C. jejuni real-time PCR assays. The mean standard curves were y = -3.36x + 43.70 and y = -3.25x + 46.20 respectively.To obtain values for the intra- and inter-assay variation of each real-time PCR assay with field samples, DNA extracted from the C. coli and C. jejuni real-time PCR assays. Indeed, for C. jejuni experimentally infected pigs, only two culture-positive samples were negative by real-time PCR, and one culture-negative sample was positive by real-time PCR (specificity of 96.2%). In addition, for pigs experimentally infected with C. coli, only one culture-negative sample was positive by real-time PCR and inversely (specificity of 96.2%).The numbers of positive and negative samples for experimentally infected pigs determined by either real-time PCR or bacteriological method are summarized in Table Campylobacter CFU/g of faeces by both C. coli and C. jejuni real-time PCR assays was compared to the bacteriological enumeration method  and for C. jejuni real-time PCR (R2 = 0.86). Among the PCR-culture positive samples for the experimentally infected pig, 72.5% of the samples had a difference in cell number of less than 1 log, 25% of less than 2 logs, and 2.5% of less than 2.5 logs for C. coli real-time PCR assay. For C. jejuni real-time PCR assay, the results obtained by real-time PCR matched equally the results obtained by culture: 67% of the samples had a difference in cell number of less than 1 log, 29% of less than 2 logs, and 4% of less than 3 logs.The estimate of d Figure . We obseC. jejuni was identified among the faecal, feed, and environmental samples from the different pig herds by conventional PCR or by our C. jejuni real-time PCR assay. Conversely, all the Campylobacter tested were identified as C. coli by both methods.No C. coli real-time PCR assay with the different field samples are reported in Table The specificity and the sensitivity for the C. coli real-time PCR matched equally the results obtained by bacterial culture: 58% of the samples had a difference in cell number of less than 1 log, 37% of less than 2 logs, and 5% of less than 3 logs.For the different field samples tested, the quantification results obtained by C. coli and C. jejuni in pure cultures and in complex samples.The real-time PCR asays developed in this study provide an effective, rapide, and sensitive alternative method to culture-based methods for the detection and the quantification of C. coli and C. jejuni real-time PCR assays allowed a precise quantification of C. coli or C. jejuni DNA amounts extracted from pure culture preparations. The specificity of the assays was assessed (i) by the species-specific amplification of DNA from different field strains/isolates of C. coli and C. jejuni, and (ii) by the absence of amplification from DNA isolated from 30 pig faecal, feed, and environmental samples previously determined to be Campylobacter-free by culture. The real-time PCR assays were also shown to be highly specific since no PCR amplicons were detected when the method was applied to DNA from different bacterial reference strains, including different Campylobacter species, Campylobacter-related bacteria, and other bacteria. Both intra- and inter-assay coefficients of variation of the Ct values for the purified genomic DNA were satisfactorily low and in concordance with those reported for other molecular assays based on PCR amplification [To use real-time PCR for quantitative measurements and to ensure a correct quantification, information on both linear range and amplification efficiency of the real-time PCR assay must be available. With a quantitative detection limit of 10 genome copies, an amplification efficiency of 99%, and a linear range of seven to eight orders of magnitude, the fication . They coC. coli and/or C. jejuni directly in pig faecal, feed, and environmental samples. In order to determine the detection limits of C. coli and C. jejuni real-time PCR assays for field samples, Campylobacter-negative faecal samples were spiked with 10-fold dilutions of the Campylobacter suspensions of each reference strain (C. jejuni NCTC 11168 and C. coli CIP 70.81). Standard curves for environmental and feed samples were constructed in a similar way. The established C. coli and C. jejuni real-time PCR assays proved highly sensitive  and were linear over a range of six orders of magnitude (from 2.0 × 102 to 2.0 × 107 CFU/g of faeces). Both intra- and inter-assay coefficients of variation of the Ct values for the DNA extracted from Campylobacter-negative faecal samples did not differ significantly. This may indicate that the main reason for variation is not due to pipetting errors in setting up the PCR assay but may be caused by contaminants from the fecal samples. Nevertheless, we did not observe systematically lower CV values of intra- and inter-assay variations with purified genomic DNA. This does not support the hypothesis that inhibitors and contaminants may interfere with uniform and consistent dilution as well as the amplification of target DNA.The technique was developed to detect and quantify Samples tested in this study constitute complex biological substrates due to the presence of (i) numerous types of bacteria, (ii) different kinds of inhibitors, and (iii) food degradation products ,37. MoreC. coli and C. jejuni real-time PCR assays respectively) leading to a sensitivity of 97.6% and 92.3%. Although the internal control was positive, we cannot exclude the hypothesis of inhibition of C. coli and C. jejuni amplification. Indeed, it was previously reported that some PCR primers are more markedly affected than others by impurities present in DNA preparations [Campylobacter spp., which has been demonstrated previously [C. jejuni signals detected by direct PCR disappeared after enrichment. Conversely C. coli signals were maintained when present before enrichment, or else became detectable when undetectable before enrichment [Campylobacter species comparatively to the other [Three faecal samples of experimentally infected pigs, detected as negative by PCR and direct streaking, were positive by culture after an enrichment step  forms or dead bacteria cells, since DNA-based tests detect all DNA of the extract from live as well as dead bacteria [Campylobacter cells in a VBNC state may potentially be still infectious [Campylobacter spp. due to differences in susceptibility to antibiotics present in selective agar [C. jejuni could be difficult due to the presence of a high background flora and due to the more numerous C. coli quantity [Furthermore, for the experimentally infected pigs, only one culture-negative faecal sample was positive by real-time PCR for each target leading to a specificity of 96.2% for both bacteria ,29,50. Ifectious ,51. The ive agar . Moreovequantity .C. coli and C. jejuni real-time PCR assays (R2 = 0.90 and R2 = 0.93 respectively). Among the PCR-culture positive samples, the real-time PCR quantification seems to be accurate compared to the culture enumeration used as a gold standard. Indeed, more than 95% of the samples with a difference in cell number of less than 2 logs, of these 72.5% and 67% less than 1 log respectively for C. coli and C. jejuni real-time PCR assays. The observed discrepancy might be due to the possible presence of VBNC forms, dead cells and antagonistic bacterial species. Another possibility could be the impact of dilution factors used for quantitative culture or an insufficient homogenization of the samples. This method provides a mean to identify and quantify at the species level C. coli and C. jejuni directly from faecal, feed, and environmental samples without requiring an enrichment step. For the different field samples tested, the qualitative data (specificity and sensitivity) as well as the quantification results obtained by C. coli real-time PCR matched equally the results obtained by bacterial culture. In this study, no C. jejuni was identified among the faecal, feed, and environmental samples from the different pig herds by conventional PCR or by our C. jejuni real-time PCR assay. Conversely, all the Campylobacter tested were identified as C. coli by both methods. In France, pigs were found to be almost always contaminated by C. coli, these first results confirmed this predominance. Nevertheless, given that we can find both species in pigs [C. coli and C. jejuni in conventional pigs.Finally, for the faecal samples of experimentally infected pigs, we observed a good correlation at the quantitative level between culture enumeration and quantitative PCR for both  in pigs ,12-14, tC. coli and C. jejuni described in this study have several advantages over culture-based techniques. These include allowing a large increase in throughput, enabling simultaneous processing of several samples , and reducing the total time required for analysis. The identification at the species level and the quantification on the entire DNA extracted from faecal, feed, and environmental samples is a new tool to enhance our understanding of the epidemiology of Campylobacter. In terms of risk assessment, this ability to differentiate and quantify these two species permits a more precise description of the carriage and excretion of C. coli and C. jejuni by livestock animals.The real-time PCR assays for Campylobacter spp., Helicobacter, Wolinella, and Arcobacter reference strains were used to test the specificity of primers and probes for real-time PCR identification and differentiation of C. coli and C. jejuni  as well as other enteric bacteria  selected from our in-house collection, the collection of the French Agency for Food, Environmental and occupational Health and Safety , and the collection of the French National Reference Center for Campylobacter and Helicobacter . Strains were stored at -80°C in brain heart infusion broth  containing 20% (v/v) glycerol. Moreover, for the real-time PCR reactions, we used the two reference strains C. jejuni NCTC 11168 and C. coli CIP 70.81 as positive controls as well as Listeria monocytogenes ATCC 19115 and Escherichia coli CIP V517 as negative controls. Campylobacter strains were grown at 25, 37 or 41.5°C for 48 h in a microaerobic atmosphere  on Karmali agar plates . Arcobacter, Helicobacter, and Wolinella were grown at 37°C for 48 h on Columbia Blood agar plates  with 5% of defibrinated sheep blood  and Enterobacter aerogenes on Purple Lactose agar plates  for 24 h. All the other bacteria listed in Table Different ni Table . In addi® Tissue mini-kit  and according to the manufacturer's instructions. The concentration of isolated double stranded DNA was determined by measuring the optical density at 260 nm with the Spectronic® Genesys™ 5 . The purity was assessed by the examination of 260/280 nm optical density ratios [i.e. having a 260/280 nm optical density ratio between 1.8 and 2.0) were adjusted to 20 ng μL-1 in TE buffer  and stored at -20°C until required for analysis.Genomic DNA from each bacterial culture was extracted using the Nucleospiny ratios . All DNAC. jejuni NCTC 11168 and C. coli CIP 70.81 cultures were extracted as described above. The genome copy numbers of C. jejuni and C. coli in 100 ng of DNA (for one PCR reaction) was calculated on the basis of the genome size  [7 and 4.6 × 107 copies respectively. After DNA quantitation by spectrofotometrical analysis with the Spectronic® Genesys™ 5, 10-fold dilutions of each extract were produced in TE buffer, representing 101 to 108 genome copies of C. jejuni per 5 μL of template (PCR reaction) and 0.3 × 101 to 3.0 × 108 genome copies of C. coli per 5 μL of template (PCR reaction). Moreover, a standard curve with roughly equal genome copies of C. jejuni and C. coli together was produced for each PCR assay. Serial DNA dilutions were aliquoted: some were stocked at 4°C to be use directly, others were stored frozen at -20°C and thawed once for use.Total genomic DNA of C. coli) -56 and wCampylobacter-negative faecal samples were obtained from specific pathogen-free (SPF) sows and piglets from the high-security barn at the Anses centre . Moreover, five Campylobacter-negative feed samples and 10 Campylobacter-negative environmental samples were collected from the same high-security barns. These samples were used to test the specificity and/or the analytical sensitivity of the real-time PCR assays. For the environmental samples, each pen of pigs was sampled on the bottom of the wall and pen partitions using swabs (sterile square pieces of cotton cloth (32 . 32 cm) moistened with isotonic saline solution) . The swabs were placed in a sterile bag before to be analyzed.Fifteen 7 CFU of Campylobacter  [n = 146). Given that pig faeces can be highly heterogeneous, rectal faecal samples were collected individually from naturally contaminated (i) sows, (ii) pigs aged three to 16 weeks, and (iii) piglets aged one to three weeks. Additionally, in the five conventional herds, 86 environmental swabs of pig pens  and 50 feed samples were collected. The swabbed surface area was measured each time.Faecal samples were obtained from both pigs experimentally inoculated with 5 × 10samples)  and natuCampylobacter spp. quantification by conventional culture and for species-identification by the PCR described by Denis et al. (1999) [All samples were examined within four hours after sampling for . (1999)  as well . (1999) .® Tissue mini-kit  with a preliminary step of boiling to remove inhibitors of the Taq polymerase [g, 5 min). For each sample, 250 μL of supernatant was extracted using the Nucleospin® Tissue mini-kit according to the manufacturer's instructions. Finally, DNA preparations, eluted in 100 μL of elution buffer purchased in the kit, were stored at +4°C prior to use.DNA isolation from the faecal, feed, and environmental samples was performed using a modified extraction protocol of the Nucleospinlymerase . Five grYersinia ruckeri was added to each sample before the DNA extraction. This internal bacterial amplification and extraction control was quantified in a separate well using a real-time PCR test described in a previous work [To test the presence of PCR inhibitors in the DNA isolated from the samples, a fixed amount of the bacterium ous work . SamplesCampylobacter numeration, 100 μL of a ten-fold dilution serie (10-1 to 10-5) were plated both on Karmali agar  and on Butzler agar  and incubated for 24 to 72 h at 41.5°C in microaerobic conditions. To promote the detection of Campylobacter in samples harbouring less than 100 Colony Forming Unit of Campylobacter per gram of faeces (CFU/g of faeces), a second plating was carried out 24 h after broth enrichment and incubated 48 h. Thermophilic Campylobacter were cultured at 41.5°C in microaerobic conditions. For direct streaking and selective enrichment, the Campylobacter suspect colonies on Karmali or Butzler plates were confirmed by microscopy (cell morphology) and conventional PCR [2 for the environmental samples were thus calculated. Finally, material from Campylobacter suspect colonies was suspended in TE buffer and subdued to DNA extraction and the species-specific PCR described by Denis et al. (1999) [C. coli and C. jejuni.Ten grams of fresh faeces, ten grams of feed, and the environmental swabs were vortexed in 90 mL of Preston broth  with a Preston antibiotic supplement  . For C. jejuni and C. coli, we have used sequences described by Lagier et al. (2004) [hipO gene (benzoylglycine amidohydrolase) responsible for the hippurate activity exclusively found within the C. jejuni genome, and (ii) on the single-copy glyA gene (serine hydroxymethyltransferase) in an unique nucleotide region within the C. coli glyA open reading frame identified as specific for C. coli [To detect . (2004) , which a C. coli   according to the recommendations of the manufacturer  probes have been designed -62. At ter Table . This so2), 400 nM of each primer , 200 nM of the probe (glyA-P and hipO-P respectively), and 5 μL of template DNA. The thermal cycle protocol used was the following: activation of the Taq DNA polymerase at 95°C for 10 min, then 45 or 48 cycles of 15 s at 95°C and 60 s at 60°C. Thermal cycling, fluorescent data collection, and data analysis were carried out with the ABI PRISM® 7300 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Fluorescence of FAM and VIC was measured at their respective wavelengths during the annealing/elongation step of each cycle. After real-time data acquisition, the baseline cycles for the FAM and VIC signals were set from cycle three to three cycles below the cycle at which the first signal appeared and the threshold value at the point at which the fluorescence exceeded 10 times the standard deviation of the mean baseline emission. The threshold cycle (Ct) is the first PCR cycle at which a statistically significant increase in fluorescent signal is detected. All reactions were carried out alongside a non template control containing all reagents except DNA, positive controls containing DNA from reference strains (C. jejuni NCTC 11168 and/or C. coli CIP 70.81), and negative controls containing DNA from Listeria monocytogenes ATCC 19115 and from Escherichia coli CIP V517. All the DNA extractions were done as described before. Each control was run in triplicate and each sample in duplicate.Reactions were done in 20 μL PCR mixtures containing 10 μL of 1X Taqman Universal PCR Mastermix , and optimised buffer components including 5 mM MgCl6 genome copies per PCR reaction) of different bacterial strains  [C. jejuni and C. coli only in field samples. Double-stranded DNA extracted from the 30 Campylobacter-negative faecal, feed, and environmental samples was examined in duplicate with each real-time PCR assay.The specificity of each real-time PCR assay was first assessed with purified genomic DNA preparations (about 10. (2004) , ensure C. jejuni NCTC 11168 and C. coli CIP 70.81, ranging from 101 to 108 genome copies per 5 μL of template (PCR reaction). In order to mimic realistic conditions and to determine the detection limits of C. coli and C. jejuni real-time PCR assays for field samples, different standard curves were prepared to quantify C. coli or C. jejuni in faecal, feed, and environmental samples. Campylobacter-negative faecal samples were spiked with 10-fold dilutions series of viable suspensions of each reference strain (C. jejuni NCTC 11168 and C. coli CIP 70.81), ranging from 101 to 108 Colony Forming Units per gram of faeces (CFU/g). Standard curves for environmental and feed samples were constructed in a similar way. DNA was extracted from each of the spiked samples and tested in real-time PCR, where the standard curves were created automatically by the ABI PRISM® 7300 Sequence Detection System Software by plotting the Ct values against each standard dilution of known concentration.The sensitivity for each PCR assay was determined using the standard curves prepared with purified genomic DNA of cultures of C. coli and C. jejuni spanning the whole range covered by each real-time PCR in different assays (10 consecutive runs) and within an assay (10 duplicates in the same assay), in order to calculate the inter- and intra-assay coefficients of variation (CV) for the Ct values experimentally determined, as previously described [The assay variability was established by repeatedly testing samples containing several concentrations of escribed .C. jejuni NCTC 11168 and C. coli CIP 70.81 from approximately 101 to 108 CFU were measured 10 times each within one PCR run. The inter-assay variation was evaluated with the same different dilutions of purified genomic DNA in 10 independent PCR experiments on different days (10 different runs). For each PCR run, each dilution point was tested in duplicate and the mean standard curve was used for quantity estimation.To assess the intra-assay variation, each dilution of purified genomic DNA of cultures from Campylobacter-negative spiked samples. To assess the intra-assay variation, DNA extracted from the Campylobacter-negative faecal samples spiked with 10-fold dilutions of the Campylobacter suspensions, ranging from 2.5 × 107 to 2.5 × 102 CFU of C. coli/g of faeces and from 2.0 × 107 to 2.0 × 102 CFU of C. jejuni/g of faeces, were measured 10 times each within one real-time PCR run. The inter-assay variation was evaluated with different dilutions of DNA extracted each time with a specific extraction from the Campylobacter-negative spiked faecal samples in 10 independent real-time PCR experiments on different days. For each real-time PCR run , each dilution point was tested in duplicate and the mean standard curves were used for quantity estimation. The CV of the Ct values were calculated for the ten different inter-assay experiments. They illustrate the variability of the Ct values obtained between experiments including the specific DNA extraction procedure and the amplification step.To assess the method with field samples, the values for the intra- and inter-assay variations of the real-time PCR assays were obtained with the DNA extracted from the C. jejuni and C. coli in pure cultures or in the field samples.The standard curves were thus used (i) to evaluate the sensitivity of the real-time PCR assays, (ii) to assess the intra- and inter-assay variabilities, and (iii) to allow a reliable quantification of (-1/slope)-1. A reaction with 100% efficiency will generate a slope of -3.32. Data analysis was performed using the SDS software (Applied Biosystems).PCR amplification efficiency (E) was estimated using the slope of the standard curve and the formula E = 10a/(a+c), where a is the number of samples found positive by both real-time PCR and bacterial culture (direct inoculation or after selective enrichment) and c is the number of samples positive by bacterial culture but negative by real-time PCR. The specificity was calculated as d/(b+d), where d is the number of samples negative by both methods and b is the number of samples positive by real-time PCR but negative by bacterial culture. Kappa-statistic was used to measure the agreement between the microaerobic cultivation and each species-specific real-time PCR assay [The 119 field samples from the experimental infection were evaluated in parallel with the real-time PCR assays and the bacterial culture described in this study. All data analyses were performed with Microsoft excel and SAS Systems version 8 . Specificity and sensitivity were assessed using the bacterial culture as a gold standard. The sensitivity was calculated as CR assay .MLM participated in the design of the study, the collection of study samples, and in the microbiological analysis; carried out the molecular genetic studies, designed the specific oligonucleotides, participated in the sequence alignment, and drafted the manuscript. MD was responsible for the experimental infection, participated in the collection and microbiological analysis of study samples, and helped to draft the manuscript. FB performed the statistical analysis, and helped to draft the manuscript. HS helped to draft the manuscript. CB participated in the study conception and coordination, provided guidance during all parts of the work, and helped to draft the manuscript. All authors read and approved the final manuscript.


Scanning electron microscopy (SEM) is a powerful tool for the direct visualization of biological specimens at nanometre-scale resolution. However, images of unstained specimens in water using an atmospheric holder exhibit very poor contrast and heavy radiation damage. Here, we present a new form of microscopy, the frequency transmission electric-field (FTE) method using SEM, that offers low radiation damage and high-contrast observation of unstained biological samples in water. The wet biological specimens are enclosed in two silicon nitride (SiN) films. The metal-coated SiN film is irradiated using a focused modulation electron beam (EB) at a low-accelerating voltage. A measurement terminal under the sample holder detects the electric-field frequency signal, which contains structural information relating to the biological specimens. Our results in very little radiation damage to the sample, and the observation image is similar to the transmission image, depending on the sample volume. Our developed method can easily be utilized for the observation of various biological specimens in water. Scanning electron microscopy (SEM) is an important technique for producing high-resolution images of biological samples Recently, we reported SEM-based methods for the analysis of biological specimens with low radiation damage Here, we present a newly developed imaging method resulting in no damage to unstained biological specimens in water, on the basis of a frequency transmission electric-field (FTE) system using SEM. Wet biological specimens are enclosed in two SiN films: the upper SiN film is coated with tungsten (W) and nickel (Ni) layers with a thickness of 20 and 5 nm, respectively. The W-Ni-coated SiN film is irradiated using a focused modulation EB at a frequency of 30−60 kHz and a low-accelerating voltage. Irradiated electrons are almost absorbed in the W layer; thus, the negative electric-field potential arises at this position. This negative potential is oscillated at the EB modulation frequency of 30−60 kHz. The electric-field oscillation is transmitted to the lower SiN film through the biological samples in water. Finally, a measurement terminal under the SiN film detects the electric frequency signal, providing structural information relating to the biological specimens. In our method, the biological samples are not directly irradiated via the EB; thus, electron radiation damage is completely avoided.A schematic of our FTE imaging method using SEM is shown in Unstained and unfixed biological specimens in water are deposited under a metal-coated SiN film and sealed using a further 50-nm-thick SiN film, which is installed in the sample holder . The samWe calculate the scattered electron distribution in a W–Ni-coated SiN film using Monte Carlo (MC) simulation . For 3-kOur detection mechanism is shown in We first observe the FTE images of unstained yeasts in a water layer sealed in the gap between two SiN films . At an ENext, we image the bubble area in the specimens . In the Finally, we measure the yeasts with environmental bacteria . At the Our FTE imaging method enables observation of unstained biological specimens in water at high contrast. Furthermore, the irradiated electrons are scattered and absorbed in the W–Ni-coated SiN film on the biological specimens; hence, our method is capable of undamaged observation. The irradiated EB is modulated by 30−60 kHz chopping and 3−4 kV acceleration . TherefoWe assume that the mechanism of propagation of the electric frequency signal at the water layer is based on the electric dipoles of the water molecules and various ions . At the The spatial resolution of our method is currently unsatisfactory at approximately 200 nm. Therefore, we are working on improving the spatial resolution. To reach 10-nm resolution, we must investigate the physical process of the signal transmission mechanism. In the future, we plan to develop a high-resolution system using high-frequency EB chopping and a 1-GHz detection amplifier. Furthermore, this system will be introduced to high-resolution field-emission SEM. We expect our future FTE system to enable the imaging of unstained biological specimens in water with a resolution greater than 10 nm, allowing observation of living viruses and macromolecular proteins.In conclusion, we have successfully imaged unstained specimens in water using a newly developed FTE system based on SEM. The specimens in the water layer are placed under the W–Ni-coated SiN film, which is sealed with another 50-nm SiN film using a sample holder. The resulting images clearly show unstained and unfixed yeasts and bacteria in water. Our method offers very low radiation damage to the sample, and the resulting image is similar to the transmission image, depending on the sample volume. Our developed method can easily be utilized for the observation of various biological specimens including living bacteria, viruses, and protein complexes in water. Furthermore, our FTE method can be used for diverse liquid samples across a broad range of scientific fields such as the nanoparticles, organic materials, and catalytic materials.2, 0.38-mm thick; Silson Ltd., UK) was coated with W and Ni using a magnetron sputter machine . The Ni sputter conditions were 1.1-Pa Ar pressure, 200-mA current, and 5-sec sputter time. The W sputter conditions were 1.1 Pa, 200 mA, and 20 sec. The distance between the sputter target and SiN films were both 50 mm. The deposited W and Ni layers were 20-nm and 5-nm thick, respectively, as shown in the cross-section image obtained by a field-emission (FE) SEM .A 50-nm-thick SiN film supported by a 0.4 mm × 0.4 mm square window in a Si frame (4 × 4 mmSacharomyces cerevisiae was obtained from DCL Yeast Ltd. (UK). Active dried yeast (10 mg) was dissolved in 1 ml of solution containing 0.5% (w/v) trehalose  and 0.5% NaCl. To ascertain the presence of yeast, a sample solution was observed by optical microscope  before preparation of the sample holder. For the yeast solution with environmental bacteria, the fresh yeasts solution was preserved at room temperature (23 °C) for one week.A yeast sample of The developed sample holder maintained the sample solution at atmospheric pressure in the sample space between a W–Ni-coated 50-nm SiN film and a 50-nm SiN film . The twoA beam-blanking unit  consisting of deflection plates was introduced into the thermionic emission SEM . The unit was controlled by a function generator  using a square wave between 0 and 10 V and 30−60 kHz frequency. The atmospheric sample holder was fixed onto an aluminium stage on the upper side of the W–Ni-coated SiN film. The sample holder with the biological specimens in water was mounted onto the sample stage, and its measurement terminal was connected to the pre-amplifier . The ele3, 8.9 g/cm3, and 3.1 g/cm3, and the thickness was 20 nm, 5 nm, and 50 nm, respectively. Physical models used for simulation were the same as in our previous study Electron trajectories in the W–Ni-coated SiN film were calculated by MC simulation using CASINO version 2.42


N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s) of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts) were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms . The sugars that are added to the protein during this process affect the physicochemical properties and polymorphism of proteins- e.g., their stabilization, protection, targeting, and direct activity –4.N-glycosylation in eukaryotes takes place along the endoplasmic reticulum (ER)-Golgi pathway, beginning with the production of a precursor. In this process, a Man5GlcNAc2 core-oligosaccharide attached to the lipid carrier dolichyl pyrophosphate (Man5GlcNAc2-PP-dolichol lipid-linked precursor intermediate) is assembled by the stepwise addition of monosaccharides to dolichol pyrophosphate on the cytosolic side of the ER [3Man9GlcNAc2-PP-dolichol lipid-linked precursor is completed [3Man9GlcNAc2 is transferred from the dolichol phosphate to the growing, nascent polypeptide chain via the nitrogen atom of an asparagine amino acid residue by Dolichyldiphosphoryloligosaccharide-protein, or oligosaccharyltransferase (OST) in the lumen of the rough ER  collectively cleave the α1,2-linked mannose residues from the oligosaccharide precursor and, thus, provide the substrates required for the formation of hybrid and complex glycans in the Golgi of eukaryotes cells. The ER members of α1,2 mannosidase in various organisms play an important role in targeting misfolded glycoproteins for degradation by proteasomes [N-glycosylation events are crucial for the proper folding of the secreted proteins and are highly conserved in the eukaryotes investigated thus far [Following the trimming of all three glucose from the Glcteasomes . ER N-glthus far .N-glycosylation patterns and N-glycan structures have been studied mainly in mammals, insects, yeasts, and plants [N-glycan structures were performed on green microalgae (Chlorophyta) [N-glycans having a xylose core residue. Whereas, two reports concerning the investigation of N-glycosylation of the green microalga Chlamydomonas reinhardtii describe two different findings [et al. 2013, revealed that the predominant N-glycans attached to Chlamydomonas reinhardtii endogenous soluble and membrane proteins, are of oligomannose type [N-linked glycans were identified as being composed of mannose, methylated mannose and xylose residues [N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii carried mammalian-like sialylated N-linked oligosaccharides [N-glycosylation pathway of the diatom Phaeodactylum tricornutum photosynthetic microalga was investigated, demonstrating that Phaeodactylum tricornutum proteins carry mainly high mannose type N-glycans (Man-5 to Man-9) and a minor glycan population carrying paucimannose type [Phaeodactylum tricornutum possesses the ER machinery required for glycoprotein quality control that is normally found in other eukaryotes [To date, d plants , with serophyta) –25. The findings ,25; Mathresidues , reportecharides . It is aose type . It was karyotes .et al. 2011 [N-linked glycans in a 66-kDa glycoprotein, which is a part of the unique sulfated complex cell wall of polysaccharide from the red microalga Porphyridium sp. N-glycans were found to be of the high-mannose type (8–9 residues), with unique modifications that included two non-characteristic xylose residues (one attached to the core and the other to the non-reducing end) and an additional methylation modification on the sixth carbon of three mannose residues attached to the chitobiose core.Red microalgae seem to have glycosylation pathways that are different from those of other known organisms, as was been concluded in a recent study by Levy-Ontman al. 2011 . This stPorphyridium sp. This organism is a photosynthetic unicell found in marine environments. One of the characteristics of red microalgae is their cell-wall that is composed of sulfated polysaccharide capsules. During growth, the external parts of the polysaccharides are released to the surrounding aqueous medium where they accumulate, increasing the medium’s viscosity [The work presented herein is focused on the red microalga iscosity –30. Thesiscosity ,32. Our iscosity –36.Porphyridium sp. [In recent years, a great deal of scientific work is being directed at creating a novel assortment of pharmaceutical products using algae as cell factories –40. Howedium sp. , the latCyanidioschyzon merolae and Galdieria sulphuraria have been completed [Porhyridium purpureum (referred to as Porphyridium cruentum) has been completed [Porhyridium purpureum genome suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life [Porphyridium sp. (sea water) and Dixoniella grisea (brakish water) [Porphyridium sp. grown under various physiological conditions generated 7210 expressed sequence tags (ESTs), which gave 2062 non-redundant sequences, containing 635 contigs and 1427 singlets [There is very limited knowledge about red algal genomes; the sequencing of genomes of the unicellular red microalgae extremophiles, Cyanidiophyceae ompleted ,49. In aompleted  and is ttic life . Moreovetic life . In addih water) ,52. Non-singlets . Some gesinglets ,52,53.N-glycosylation mechanism that takes place in the ER within the red microalga Porphyridium sp. Our DNA scaffold (SCF) database of Porphyridium sp. was used to search for sequence similarity to algae gene products potentially involved in N-glycosylation pathways. Such a study can provide a basis for understanding N-glycosyation pathways in red microalgae, and lay the foundations for future gene cloning and characterization.In this paper we describe our attempt to better understand the 2.2.1.DNA was divided into sections of 330 bases (on average) and 38 bases were sequenced from each end of each section (Pair-end). The total reads identified were 38,537,782 sections, constituted of 1,464,435,716 bases.i.e., less spurious overlaps), but lower coverage. A k-mer of 23 indicates more specificity on account of coverage. Nevertheless, we were able to obtain an impressive length of contigs, with N50 of 41,031 bp for the SCF , Phaeodatylum tricornutum (genome size 27.4 MB), the green algae Ostrecoccus tauri (genome size 12.6 MB), Ostrecoccus lucimarinus (genome size 13.2 MB), and Micromonas pussila (genome size 21 MB) [Assembly of all reads was completed using VELVET; the best assembly results of the reads was obtained with a hash (or k-mer) of 23. Longer k-mers bestow more specificity ( the SCF . The ass the SCF : (1) cone 21 MB) .2.2.N-glycosylation pathway were conducted by using the TBLASTN function on the algae DNA SCF contigs engine database (Section 3.3.3). TBLASTN was selected because there is very little evidence for introns in Porphyridium sp. . In order to identify Porphyridium sp. gene products involved in the ER N-glycosylation pathway, homology-based searches of Saccharomyces cerevisiae (S. cerevisiae) N-glycosylation genes against the Porphyridium sp. DNA SCF contigs engine were conducted. The identification of the calreticulin encoding-gene in Porphyridium sp. was based on homology-based searches against the Chlamydomonas reinhardtii ortholog gene, and that of UGGT encoding-gene was based against Galdieria sulphuraria ortholog gene. Searches for encoding-genes of OST 3/6/5 and SWP1 were based on homology-based searches against the ortholog genes in S. cerevisiae, Chlamydomonas reinhardtii, Galdieria sulphuraria, Cyanidioschyzon merolae and Arabidopsis thaliana. Homologs were found for almost all algal N-glycosylation protein sequences in the ER pathway because all of them exhibit similarity values of above 43% under good sequence coverage calculated as compared to the entire gene sequences (above 60%), with one exception (sequence coverage of UGGT was only 26%) (N-glycosylation pathway homologues (Homology searches based on sequence similarities with genes encoding proteins involved in ER nly 26%) . In addimologues . The hommologues .Porphyridium sp. , accounts alone for OST activity in some organisms [Porphyridium sp. functions without OST5, OST3/6, and SWP1 subunits. It is also important to note that we identified two STT3 copies in the Porphyridium sp. genome .Taken together, these results suggest that the ER 2.3.N-glycosylation pathway of Porphyridium sp. and ortholog protein sequences of other organisms. The various organisms tested included red microalgae , green microalgae , diatoms , mammals , and the yeast S. cerevisiae. It appears that most of the genes involved in the ER N-Glycosylation pathway in Porphyridium sp. also exist in other red algae, green algae, diatoms, yeasts, and mammals  to the N-glycan and GCS1 is responsible for trimming the Glc α(1–2) residue after the N-glycan is transferred to the nascent protein. This assumption was also verified by comparing between the similarity of the Porphyridium sp. STT3 sequence to ortholog genes of organisms that did or did not contain the ALG10 and GCS1 genes. Since the subunit STT3 is accepted as a substrate of the ALG10 enzyme, paucity in glucose residues on the substrate can change the connection to STT3. Indeed, the similarity between Porphyridium sp. STTs gene to organisms that were found to have ALG10 and GCS1 was higher compared to organisms that lacked the ALG10 and GCS1, indicated by changes in STT3 subunit affinity.TBLASTN, a bioinformatics-based similarity tool, was used to compare between protein sequences involving the ER  mammals . Most ofrganisms . It is nthaliana . Looking diatoms . The genPorphyridium sp. encoded-genes to ortholog genes of complex eukaryotes, and the resemblance of Porpyridium sp. OST complex to that found in lower organisms (including prokaryotes) it appears that the red alga retained genes from both partners, thus, bringing together mutual elements in the red alga genome. Indeed, the N-glycan structures of the cell-wall glycoprotein within the Porpyridium sp. polysaccharide were also found to be composed of prokaryote to multicellular organism elements [Based on the strong similarity of elements .N-glycosylation enzymes of Porphyridium sp.  was compared to ortholog parts of other organisms—other algae, plant and non-photosynthesic organisms—and a phylogenetic analysis was performed , obtained from University of Texas Culture Collection, was grown in 250 mL Erlenmeyer flasks, each containing 100 mL of artificial seawater (ASW) [2. The growth temperature was maintained at 25 ± 3 ºC, and illumination was supplied continuously from above with white fluorescent lamps at a photon flux density of 90 μmol photons m−2s−1. Cells were counted with a haemocytometer using a Zeiss light microscope .The microalga er (ASW) . The alg3.2.3.2.1.6 cells/mL) was centrifuged  and the pellet was washed with acidified water (pH 4) to remove cell-bound polysaccharides and then centrifuged . The remaining pellet (~1 g) was frozen immediately in liquid-nitrogen and minced into powder with a mortar and pestle. The pellet was incubated with gentle rocking for 2 h at 65 ºC. Ten milliliters of CTAB buffer (CTAB—3% w/v), NaCl—1.4 M, EDTA (pH = 8)—20 mM, Tris-HCl (pH = 8)—10 mM, 0.1% β-Mercaptoehanol) were then added to the incubated pellet. The mixture was extracted by mixing it with a 10 mL solution of chloroform: isoamyl alcohol (24: 1 v/v) and then incubating the mixture for 5 min at 25 ºC. Later, the extraction mixture was centrifuged  and the supernatant was collected to a clean vial. The extraction procedure was repeated 3 times. DNA precipitation was obtained by adding isopropyl alcohol  to the collected supernatant, mixing gently and incubating at −20 ºC for 20 min, until a precipitate was formed. The DNA precipitate was collected by centrifuging , washing in 70% ethanol, drying by vacuum centrifuge and resuspending in 300 μL DDW. To remove residual RNA, the sample was incubated with RNaseA , for 1.5 h at 37 ºC. Afterwards the sample was precipitated again by adding 3 M sodium acetate pH 5.2  and cold absolute ethanol . The sample was centrifuged , and the resulting preciptate was washed in 70% ethanol, dried by vacuum centrifuge, and resuspended in 300 μL DDW. DNA concentration was determined by nanodrop and gel electrophoresis.A 48-h logarithmic-scale cell culture  was sequenced at Fasteris, Switzerland by the high-throughput sequencing method of Solexa technology with Illumina’s Genome Analyzer EAS210R version-GA IIx  using the Pair-end method.DNA extracted from 3.2.3.de novo assembly [The assembly of the reads to contigs was done using VELVET Version 0.7.54 suitable for assembly . Validatassembly .In order to receive two database sources, two types of assemblies were made: (1) contigs database source containing DNA sequences based solely on reads, and (2) SCF database source, made by close contigs that were adjacent to each other using a number of unknown bases (N’s).3.3.Based on the fact that most algal genes have no introns, the partitioning of the sequences to ORFs was programmed with Glimmer v 3.02 , in the 3.3.1.Analysis and annotation of the genes and proteins were done by blast2go ,84.3.3.2.The DNA sequences were translated into protein sequences by a program available on the ExPASy  .3.3.3.Porphyridium sp. SCF sequences database (Section 3.2.3), a search engine based on homological sequences according to similarity, was created by BLAST using Blast Command Line Application program [Based on the  program , which i3.4.S. cerevisiae/Chlamydomonas reinhardtii/Galdieria sulphuraria/Cyanidioschyzon merolae/ Arabidopsis thaliana were used as a reference species for sequence-based comparative analysis. Sequences belonging to the N-glycosylation pathway of these reference species were taken from Gene bank (NCBI) and used for searching for homological sequences according to similarity in the sequences of the algae’s DNA using the SCF search engine (Section 3.3.3). The sequences identified in this study were translated into protein sequences (Section 3.3.2) in order to set the start and end of each protein sequence . Each sequence was analyzed by BlastP to ensure the appropriate annotation to the related N-glycosylation proteins. In addition, multiple alignments were performed using the ClustalW algorithm [lgorithm .3.5.N-glycosylation proteins based on the algae genome was conducted by blast2go. Separately, sequence detection of conserved domains was performed directly by Interpro and the added GO’s (Gene ontology) [A homology study of conserved domains in various related ntology) –91, whic3.6.N-glycosylation pathway of various organisms were taken from Gene bank (NCBI) and used for comparative similarity with the predicted ortholog sequences of the red microalga Porphyridium sp. The results were achieved using BLAST 2 seq [Sequences belonging to encoded-genes involved in the ST 2 seq ,84.3.7.N-glycosylation, was built based on integration of three major enzyme sequences involving Porphyridium sp. N-glycosylation pathway: ALG1, ALG7, and GANAB, and closely related enzymes from different organisms based on amino acid sequences obtained from NCBI protein database. The conserved domains of Porphridium sp. sequences were detected by Interposcan. The tree was built using the Neighbor-Joining method with 500 repeated bootstrap tests, using MEGA software, version 4.0 [A Phylogenetic tree showing evolutionary relationships of enzymes involved in sion 4.0 .4.N-glycosylation pathway that occurs in the ER involves a large number of proteins, the majority of which are catalytic enzymes that are relatively well conserved in different organisms. We have identified genes encoding 24 products implicated in the ER N-glycan biosynthesis in the red microalga Porphyridium sp. A bio-informatic analysis of the Porphyridium sp. genome revealed the presence of a complete set of sequences potentially encoding for the following proteins: those involved in the synthesis of the lipid-linked Glc3Man9GlcNAc2-PP-dolichol N-glycan; some subunits of the OST complex; ER glucosidases and chaperones required for protein quality control; and finally, the ER α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. As all the encoded genes involved in eukaryote N-glycosylation (before the N-glycan is transferred to the protein) were found in the Porphyridium sp. genome, it can be assumed that the final structure of the N-glycan in the ER before it is transferred to the protein, is the usual precursor Glc3Man9GlcNAc2 as known in other organisms.The N-glycosylation pathway in Porphyridium sp. was also undertaken. The similarity of protein sequences to ortholog sequences from red and green algae, diatoms, mammals, and yeasts was studied. The similarity was found to be relatively high (above 40%), indicating the degree of conservation of the N-glycosylation pathway, and its importance in eukaryotes in general, and photosynthetic organisms in particular. This study showed that the genomes of several species of green algae and all the diatoms lack two enzyme sequences, ALG10 and GCS1. Furthermore, it was found that the similarity of the STT3 gene of these organisms  in relation to Porphyridium sp. ortholog gene was smaller in comparison to the other organisms that were tested. As the ALG10 product is a substrate for STT3, it is likely that these organisms  do not express the STT3 products as active enzymes. These findings indicate a close evolutionary relation of red algae to complex eukaryotes. Conversely, the OST encoded-subunits that were missing  and the existence of several copies of the STT3 gene in Porphyridium sp. indicate its relation to lower organisms such as diatoms. This finding supports the theory that endosymbiosis took place between a eukaryote and a cyanobacterium as a single event that gave rise to all photosynthetic organisms. In addition, the grouping of Porphyridium sp. sequences with those of other red algae confirms that the homologs found in this study are in fact orthologs of the N-glycosylation enzymes that are also found in eukaryotes.A study of gene conservation in the ER Porphyridium sp. contains the majority of encoded-genes responsible for the N-glycosylation pathway in the ER as in eukaryotes organisms. Studies to elucidate the exact mode of action of these encoded-gene products are currently under way.In summary, we demonstrated that